This application represents a continuation of the Principal Investigator's studies on the expression of the factor IX (FIX) gene. In previous studies, he has shown that an intron sequence as well as a silencer element located at the 5' end of the gene and a small 5' region called LS are involved in the developmental regulation of this gene. The proposed studies will more clearly define these sequences using both deletion and site-specific mutagenesis. In addition, gel mobility and footprinting studies will be used to describe the interaction between regulatory proteins and the DNA. These proteins will then be isolated and characterized. The initial studies will be carried out using cell cultures but as the studies proceed, transgenic mouse assays will be utilized to determine if the regulation including the identified cis-acting elements and trans-acting factors are operable in whole animals. In these studies, a mini-gene construct will be utilized. The Principal Investigator has also proposed a novel approach for studying regulation of the mutant factor IX genes such as FIX-Leyden. One difficulty is that these genes are expressed in the liver, however, obtaining such samples from patients is difficult if not impossible. Therefore, it is proposed that lymphocytes from patients, which carry the altered gene, be fused to hepatoma cell lines to both immortalize the cells and provide liver specific expression. In this way, it may be possible to study in detail, the expression of the altered genes. Finally, the FVII gene will be studied using the same approach used to characterize the FIX gene.